Methods of modulating of angiogenesis

ABSTRACT

A method of inhibiting angiogenesis in a mammal by administering to the mammal a compound which inhibits binding of endothelial PAS domain protein-1 to cis-acting transcription regulatory sequence in the promoter region of a gene encoding an angiogenic factor.

BACKGROUND OF THE INVENTION

[0001] This invention relates to vascular therapy.

[0002] Angiogenesis results from endothelial cell proliferation inducedby angiogenic factors. Angiogenic factors bind to receptors onendothelial cells which line blood vessels. This event triggers signalswhich cause the cells to proliferate; the proliferating endothelialcells secrete proteases which digest the basement membrane surrounding avessel. The junctions between the endothelial cells are altered,allowing projections from the cells to pass through the space created.These outgrowths then become new blood vessels, e.g., capillaries.

[0003] Vascular endothelial cell growth factor (VEGF) and VEGF receptors(VEGF-Rs) play a role in vasculogenesis and angiogenesis. Although VEGFis secreted by a variety of cell types, including vascular smooth musclecells, osteoblasts, fibroblasts, and macrophages, its proliferative andchemotactic activities are restricted to endothelial cells. VEGFsignaling is mediated by two VEGF-Rs, the endothelial cell-specifictyrosine kinase receptors, flt-1 and KDR/flk-1. Despite its importancein VEGF signaling, the molecular mechanisms of VEGF and VEGF-Rexpression have not been elucidated.

SUMMARY OF THE INVENTION

[0004] The invention is based on the discovery that endothelial PASdomain protein-1 (EPAS1) binds to cis-acting regulatory sequencesassociated with genes encoding such angiogenic factors as VEGF andVEGF-Rs such as KDR/flk-1 and flt-1, thereby transactivating thepromoters of such genes. Accordingly, the invention features a method ofincreasing the level of EPAS1 in a cell, e.g., an endothelial cell. Anincrease in the level of EPAS1 leads to increased promotertransactivation and increased transcription of genes encoding angiogenicfactors which participate in the blood vessel formation.

[0005] The invention also includes a novel basichelix-loop-helix/Per-AhR-Arnt-Sim (bHLH/PAS) protein which binds toEPAS1 and forms a heterodimer which transactivates transcription ofgenes encoding angiogenic factors. Increasing the level of ARNT4 in acell, e.g., an endothelial cell also leads to increased promotertransactivation and increased expression of angiogenic factors whichparticipate in the blood vessel formation.

[0006] Angiogenic factors are proteins or polypeptides and ligandsthereof that participate in the process of new blood vessel formation.For example, angiogenic factors include VEGF, VEGF-Rs, and othersignalling proteins such as intracellular tyrosine kinases whichparticipate in the angiogenic process. Preferably, the angiogenicfactors are expressed in endothelial cells, e.g., VEGF, VEGF-Rs such asKDR/flk-1 or flt-1, and tyrosine kinases such as Tie2.

[0007] A method of inhibiting angiogenesis in a mammal is carried out byadministering to the mammal a compound which inhibits binding of EPAS1to cis-acting transcription regulatory DNA associated with a geneencoding an angiogenic factor. Angiogenesis is also inhibited byadministering a compound which inhibits binding of EPAS1 to ARNT4, i.e.,a compound which inhibits the formation of a functional heterodimer thatcan transactivate a promoter of gene encoding an angiogenic factor. Theangiogenic factor is preferably VEGF., a VEGF-R such as KDR/flk-1 orflt-1. For example, the compound inhibits transcription of theangiogenic factor by binding to a cis-acting regulatory sequence such asthe sequence 5′GCCCTACGTGCTGTCTCA 3′ (SEQ ID NO:1) in VEGF promoter DNA.For example, the compound is an EPAS1 polypeptide that binds to acis-acting regulatory sequence of a gene but fails to transactivate thepromoter of the gene, e.g. a polypeptide lacking a transactivationdomain (amino acids 486-690 of EPAS1). TABLE 1 Transactivation domain ofhuman EPAS1 EDYYTSLDNDLKIEVIEKLFAMDTEAKDQCSTQTDFNELDLETLAPYIPMDGEDFQLSPI(SEQ ID NO:2)CPEERLLAENPQSTPQHCFSAMTNIFQPLAPVAPHSPFLLDKFQQQLESKKTEPEHRPMSSIFFDAGSKASLPPCCGQASTPLSSMGGRSNTQWPPDPPLHFGPTKWAVGDQRTEFLGAAPLGPPVSPPHVSTFKTRSAKGFGAR

[0008] When such an EPAS1 mutant is bound to a cis-acting regulatoryDNA, it prevents wild type EPAS1 binding and thereby inhibitstranscription of a gene encoding an angiogenic factor (and, in turn,angiogenesis). For example, the EPAS1 polypeptide contains theN-terminal binding domain (amino acids 14-67 of EPAS1;RRKEKSRDAARCRRSKETEVFYELAHELPLPHSVSSHLDKASIMRLEISFLRTH; SEQ ID NO: 3),more preferably the EPAS polypeptide contains amino acids 1-485 of humanEPAS1. The amino acid sequence of such an EPAS1 dominant negative mutantpolypeptide and DNA encoding such a mutant polypeptide is providedbelow. TABLE 2 EPAS1 dominant negative mutant 1 MTADKEKKRS SSERRKEKSRDAARCRRSKE TEVFYELAHE LPLPHSVSSH 51 LDKASIMRLE ISFLRTHKLL SSVCSENESEAEADQQMDNL YLKALEGFIA 101 VVTQDGDMIF LSENISKFMG LTQVELTGHS IFDFTHPCDHEEIRENLSLK 151 NGSGFGKKSK DMSTERDFFM RMKCTVTNRG RTVNLKSATW KVLHCTGQVK201 VYNNCPPHNS LCGYKEPLLS CLIIMCEPIQ HPSHMDIPLD SKTFLSRHSM 251DMKFTYCDDR ITELIGYHPE ELLGRSAYEF YHALDSENMT KSHQNLCTKG 301 QVVSGQYRMLAKHGGYVWLE TQGTVIYNPR NLQPQCIMCV NYVLSEIEKN 351 DVVFSMDQTE SLFKPHLMAMNSIFDSSGKG AVSEKSNFLF TKLKEEPEEL 401 AQLAPTPGDA IISLDFGNQN FEESSAYGKAILPPSQPWAT ELRSHSTQSE

[0009] 451 AGSLPAFTVP QAAAPGSTTP SATSSSSSCS TPNSP (SEQ ID NO: 4) TABLE 3DNA encoding EPAS1 Dominant Negative Mutantcctgactgcgcggggcgctcgggacctgcgcgcacctcggaccttcaccacccgcccggg (SEQ IDNO:5) ccgcggggagcggacgagggccacagccccccacccgccagggagcccaggtgctcggcgtctgaacgtctcaaagggccacagcgacaatgacagctgacaaggagaagaaaaggagtagctcggagaggaggaaggagaagtcccgggatgctgcgcggtgccggcggagcaaggagacggaggtgttctatgagctggcccatgagctgcctctgccccacagtgtgagctcccatctggacaaggcctccatcatgcgactggaaatcagcttcctgcgaacacacaagctcctctcctcagtttgctctgaaaacgagtccgaagccgaagctgaccagcagatggacaacttgtacctgaaagccttggagggtttcattgccgtggtgacccaagatggcgacatgatctttctgtcagaaaacatcagcaagttcatgggacttacacaggtggagctaacaggacatagtatctttgacttcactcatccctgcgaccatgaggagattcgtgagaacctgagtctcaaaaatggctctggttttgggaaaaaaagcaaagacatgtccacagagcgggacttcttcatgaggatgaagtgcacggtcaccaacagaggccgtactgtcaacctcaagtcagccacctggaaggtcttgcactgcacgggccaggtgaaagtctacaacaactgccctcctcacaatagtctgtgtggctacaaggagcccctgctgtcctgcctcatcatcatgtgtgaaccaatccagcacccatcccacatggacatccccctggatagcaagaccttcctgagccgccacagcatggacatgaagttcacctactgtgatgacagaatcacagaactgattggttaccaccctgaggagctgcttggccgctcagcctatgaattctaccatgcgctagactccgagaacatgaccaagagtcaccagaacttgtgcaccaagggtcaggtagtaagtggccagtaccggatgctcgcaaagcatgggggctacgtgtggctggagacccaggggacggtcatctacaaccctcgcaacctgcagccccagtgcatcatgtgtgtcaactacgtcctgagtgagattgagaagaatgacgtggtgttctccatggaccagactgaatccctgttcaagccccacctgatggccatgaacagcatctttgatagcagtggcaagggggctgtgtctgagaagagtaacttcctattcaccaagctaaaggaggagcccgaggagctggcccagctggctcccaccccaggagacgccatcatctctctggatttcgggaatcagaacttcgaggagtcctcagcctatggcaaggccatcctgcccccgagccagccatgggccacggagttgaggagccacagcacccagagcgaggctgggagcctgcctgccttcaccgtgccccaggcagctgccccgggcagcaccacccccagtgccaccagcagcagcagcagctgctccacgcccaatagcccttga

[0010] Rather than administering EPAS1 polypeptides or ARNT4polypeptides, the method may be carried out by administering DNAencoding such polypeptides. For example, the compound is a nucleic acidencoding an EPAS1 polypeptide lacking amino acids 486-690 of EPAS1. Forexample, the nucleic acid encodes a dominant negative mutant of EPAS1which contains amino acids 1-485 of wild type EPAS1, i.e., SEQ ID NO: 5.

[0011] For antisense therapy, the compound is a antisense nucleic acidmolecule containing at least 10 nucleotides the sequence of which iscomplementary to an mRNA encoding all or part of a wild type EPAS1polypeptide. Preferably, the compound, e.g., an antisenseoligonucleotide or antisense RNA produced from an antisense template,inhibits EPAS1 expression. For example, the compound may inhibit EPAS1expression by inhibiting translation of EPAS1 mRNA. For example,antisense therapy is carried out by administering a single strandednucleic acid complementary at least a portion of EPASI MRNA to interferewith the translation of MRNA into protein, thus reducing the amount offunctional EPAS1 produced in the cell. A reduction in the amount offunctional transactivating EPAS1 reduces the level of transcription ofangiogenic factors such as VEGF or VEGF-Rs, resulting in a decrease innew blood vessel formation.

[0012] Alternatively, the compound is an EPASI-specific intrabody, i.e.,a recombinant single chain EPAS1-specific antibody that is expressedinside a target cell, e.g., a vascular endothelial cell. Such anintrabody binds to endogenous intracellular EPAS1 and prevents it frombinding to its target cis-acting regulatory sequence in the promoterregion of a gene encoding an angiogenic factor such as VEGF or a VEGF-R.An ARNT4-specific intrabody is also useful to inhibit angiogenesis.

[0013] Angiogenesis contributes to the progression of atheroscleroticlesions. Thus, compounds are administered to a site of anatherosclerotic lesion in a mammal to inhibit growth of a lesion.Compounds may also be locally administered to a tumor site to reduceblood vessel formation, thereby depriving a tumor of blood supply andinhibiting tumor growth. VEGF itself is a growth factor for some tumors;the methods described above directly inhibit VEGF expression, and thus,are particularly useful for treating such tumor types.

[0014] The invention also includes an antibody which binds to EPAS1. Theantibody preferably binds to the C-terminal portion of EPAS1 (e.g., apolypeptide having the amino acid of SEQ ID NO: 17 or 18). The antibodyis a polyclonal or monoclonal antibody which specifically binds to theEPAS1. Preferably, the antibody binds to an epitope within theC-terminal transactivation domain (SEQ ID NO: 2). The inventionencompasses not only an intact monoclonal antibody, but also animmunologically-active antibody fragment, e.g., a Fab or (Fab)₂fragment; an engineered single chain Fv molecule; or a chimericmolecule, e.g., an antibody which contains the binding specificity ofone antibody, e.g., of murine origin, and the remaining portions ofanother antibody, e.g., of human origin.

[0015] To promote angiogenesis in a mammal, a compound, e.g., DNAencoding EPASI or a functional fragment thereof, which increasesexpression of VEGF or a VEGF-R in an endothelial cell is administered toa mammal, e.g., an adult mammal which has been identified as being inneed of therapy to promote angiogenesis such as a patient suffering fromperipheral vascular disease. A functional fragment of EPAS1 is one whichbinds to DNA in the promoter region of a gene encoding an angiogenicfactor.

[0016] The invention also features an EPAS-binding element, ARNT4 and anucleic acid which encodes ARNT4. For example, the nucleic acid includesa sequence which encodes the amino acid sequence a naturally-occurringhuman ARNT4 (SEQ ID NO: 19). The DNA may encode a naturally-occurringmammalian ARNT4 polypeptide such as a human, rat, mouse, guinea pig,hamster, dog, cat, pig, cow, goat, sheep, horse, monkey, or ape ARNT4.Preferably, the DNA encodes a human ARNT4 polypeptide, e.g., apolypeptide which contains part or all of the amino acid sequence of SEQID NO: 19. The invention includes degenerate variants of the human cDNA(SEQ ID NO: 20) . The DNA contains a nucleotide sequence having at least50% sequence identity to SEQ ID NO: 20. For example, the DNA contains asequence which encodes a human ARNT4 polypeptide, such as the codingsequence of SEQ ID NO: 20 (nucleotides 220 to 2025 of SEQ ID NO: 20).The DNA contains a strand which hybridizes at high stringency to astrand of DNA having the sequence of SEQ ID NO: 20, or the complementthereof. The DNA has at least 50% sequence identity to SEQ ID NO: 20 andencodes a polypeptide having the biological activity of a ARNT4polypeptide, e.g, the ability to bind to EPAS1 to form a heterodimer.Preferably, the DNA has at least 75% identity, more preferably 85%identity, more referably 90% identity, more preferably 95% identity,more preferably 99% identity, and most preferably 100% identity to thecoding sequence of SEQ ID NO: 20.

[0017] Nucleotide and amino acid comparisons are carried out using theCLUSTAL W sequence alignment system with (Thompson et al., 1994, NucleicAcids Research 22:4673-4680 orhttp://www.infobiogen.fr/docs/ClustalW/clustalw.html). Amino acidsequences were compared using CLUSTAL W with the PAM250 residue weighttable. “Per cent sequence identity”, as that term is used herein, isdetermined using the CLUSTAL W sequence alignment system referencedabove, with the parameters described herein. In the case of polypeptidesequences which are less than 100% identical to a reference sequence,the non-identical positions are preferably, but not necessarily,conservative substitutions for the reference sequence. Conservativesubstitutions typically include substitutions within the followinggroups: glycine and alanine; valine, isoleucine, and leucine; asparticacid and glutamic acid; asparagine and glutamine; serine and threonine;lysine and arginine; and phenylalanine and tyrosine.

[0018] Hybridization is carried out using standard techniques, such asthose described in Ausubel et al. (Current Protocols in MolecularBiology, John Wiley & Sons, 1989). “High stringency” refers to nucleicacid hybridization and wash conditions characterized by high temperatureand low salt concentration: wash conditions of 65° C. at a saltconcentration of 0.1×SSC. “Low” to “moderate” stringency denotes DNAhybridization and wash conditions characterized by low temperature andhigh salt concentration: wash conditions of less than 60° C. at a saltconcentration of 1.0×SSC. For example, high stringency conditionsinclude hybridization at 42° C., and 50% formamide; a first wash at 65°C., 2×SSC, and 1% SDS; followed by a second wash at 65° C. and 0.1%×SSC.Lower stringency conditions suitable for detecting DNA sequences havingabout 50% sequence identity to an ARNT4 gene are detected by, forexample, hybridization at 42° C. in the absence of formamide; a firstwash at 42° C., 6×SSC, and 1% SDS; and a second wash at 50° C., 6×SSC,and 1% SDS.

[0019] A vector containing an ARNT4-encoding DNA is also within theinvention. Preferably the DNA which includes an ARNT4-encoding DNA isless than 5 kilobases in length; more preferably, the DNA is less than 4kilobases in length, more preferably the DNA is less than 3 kilobases inlength, and most preferably the DNA is approximately 2 kilobases or lessin length. The invention also provides a method of directingcardiac-specific or smooth muscle cell-specific expression of a proteinby introducing into a cell an isolated DNA containing a sequenceencoding the protein operably linked to the tissue-specific promoter. Acell containing the DNA or vector of the invention is also within theinvention.

[0020] By “substantially pure DNA” is meant DNA that has anaturally-occurring sequence or that is free of the genes which, in thenaturally-occurring genome of the organism from which the DNA of theinvention is derived, flank the ARNT4 gene. The term therefore includes,for example, a recombinant DNA which is incorporated into a vector, intoan autonomously replicating plasmid or virus, or into the genomic DNA ofa procaryote or eucaryote at a site other than its natural site; orwhich exists as a separate molecule (e.g., a cDNA or a genomic or cDNAfragment produced by PCR or restriction endonuclease digestion)independent of other sequences. It also includes a recombinant DNA whichis part of a hybrid gene encoding additional polypeptide sequence.

[0021] Also within the invention is a substantially pure human ARNT4polypeptide. The term ARNT4 polypeptide includes a polypeptide havingthe amino acid sequence and length of the naturally-occurring ARNT4 aswell as fragments of the full-length naturally-occurring ARNT4. Thepolypeptide contains the amino acid sequence of SEQ ID NO: 19.Preferably the polypeptide contains an amino acid sequence which is atleast 50% identical to SEQ ID NO: 19. Preferably, the amino acidsequence has at least 75% identity, more preferably 85% identity, morepreferably 90% identity, more preferably 95% identity, more preferably99% identity, and most preferably 100% identity to the amino acidsequence of SEQ ID NO: 19. For example, the ARNT4 polypeptide may havethe amino acid sequence of the naturally-occurring human polypeptide,e.g., a polypeptide which includes the amino acid sequence of SEQ ID NO:19. The invention also encompasses a polypeptide with the amino acidsequence of a segment of SEQ ID NO: 17 which spans residues 75 to 128,inclusive, or a segment spanning residues 155 to 207, inclusive, of SEQID NO: 19, or a segment spanning residues 232 to 384 of SEQ ID NO: 19.Preferably, such a polypeptide has a biological activity of anaturally-occurring ARNT4 polypeptide, e.g, heterodimer formation withEPAS1 or the ability to transactivate transcription under the control ofa VEGF promoter.

[0022] A substantially pure ARNT4 polypeptide is obtained by extractionfrom a natural source; by expression of a recombinant nucleic acidencoding a ARNT4 polypeptide; or by chemically synthesizing the protein.A polypeptide or protein is substantially pure when it is separated fromthose contaminants which accompany it in its natural state (proteins andother naturally-occurring organic molecules). Typically, the polypeptideis substantially pure when it constitutes at least 60%, by weight, ofthe protein in the preparation. Preferably, the protein in thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight, ARNT4. Purity is measured by anyappropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis. Accordingly, substantially purepolypeptides include recombinant polypeptides derived from a eucaryotebut produced in E. coli or another procaryote, or in a eucaryote otherthan that from which the polypeptide was originally derived.

[0023] The invention also includes a transgenic non-human mammal, thegerm cells and somatic cells of which contain a null mutation in a geneencoding an ARNT4 polypeptide. For example, the null mutation is adeletion of part or all of an exon of ARNT4. Preferably, the mammal is arodent such as a mouse. An antibody which specifically binds to a ARNT4polypeptide is also within the invention.

[0024] Angiogenesis is inhibited by administering to a mammal a compoundwhich inhibits binding of EPAS1 to ARNT4 such as an ARNT4 polypeptide.For example, the compound is a polypeptide or peptide mimetic whichcontains the amino acid sequence of residues 75 to 128, inclusive, ofSEQ ID NO: 19, the amino acid sequence of residues 155 to 207,inclusive, of SEQ ID NO: 19, or a the amino acid sequence of residues232 to 384 of SEQ ID NO: 19.

[0025] Other features and advantages of the invention will be apparentfrom the following detailed description and from the claims.

DETAILED DESCRIPTION

[0026] The drawings will first be described.

DRAWINGS

[0027]FIG. 1A is a bar graph showing dose-dependent transactivation ofKDR/flk-1 promoter by EPAS1. EPAS1 expression plasmid phEP-1 (0-6 μg),pcDNA3 (6-0 μg), and KDR/flk-1 reporter pGL2-4kb+296 (1 μg) weretransfected into BAEC.

[0028]FIG. 1B is a bar graph showing that deletion of EPAS1 C-terminalregion abolishes its ability to transactivate the KDR/flk-1 promoter.Expression plasmids (6 μg each) and pGL2-4.Okb+296 (1 μg) werecotransfected into BAEC. For all constructs in FIGS. 1A-B, the plasmidpCMV-βGAL was cotransfected to correct for differences in transfectionefficiency. In both FIGS. 1A and 1B, luciferase activity andβ-galactosidase activity were measured, and normalized luciferaseactivity was calculated as described below. The “fold induction”represents the ratio (mean±SE) of normalized luciferase activity incells transfected with expression plasmid to that in cells transfectedwith empty vector (pcDNA3).

[0029]FIG. 2A is a bar graph showing transactivation of the KDR/flk-1promoter by EPAS1 but not by HIF-1a (another member of the PAS family oftranscription factors). Expression plasmids (6 μg each) and KDR/flk-1reporter pGL2-4kb+296 (1 μg) were cotransfected into the cell typesindicated.

[0030]FIG. 2B is a bar graph showing transactivation of a VEGF promoterby EPAS1 and HIF-1α. Expression plasmids (6 μg each) and VEGF reporterpVR47/CAT (1 μg) were cotransfected into the cell types indicated. Forall constructs in FIGS. 2A-2B, the plasmid pCMV- GAL was cotransfectedto correct for differences in transfection efficiency. The “foldinduction” represents the ratio (mean+SE) of normalized luciferase orCAT activity in cells transfected with expression plasmid to that incells transfected with empty vector (pcDNA3).

[0031]FIG. 3A is a diagram showing an alignment of the amino acidsequence of human ARNT4 with human BMALlb and human ARNT.

[0032]FIG. 3B is a diagram of a phylogenetic tree of the ARNT family ofproteins.

[0033]FIG. 4 is a bar graph showing the results of a yeast two-hybridassay. ARNT3 (BMALlb) and ARNT4 form heterodimers with EPAS1 as well aswith CLOCK.

[0034]FIG. 5 is a bar graph showing that EPAS1 interacts with ARNT4 toform functional heterodimers which increase VEGF promoter activity andVEGF expression.

[0035]FIG. 6 is a bar graph showing that ARNT4 does not interact withHIF-1α.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] EPAS1 is a member of the transcription factor familycharacterized by a basic helix-loop-helix (bHLH) domain and a(Per-AhR-Arnt-Sim) PAS domain composed of two imperfect repeats. Table 4shows the amino acid sequence of human wild type EPAS1. TABLE 4 Aminoacid sequence of human EPAS1MTADKEKKRSSSERRKEKSRDAARCRRSKETEVFYELAHELPLPHSVSSHLDKASIMRLE (SEQ IDNO:6) ISFLRTHKLLSSVCSENESEAEADQQMDNLKALEGFIAVVTQDGDMIFLSENISKFMGLTQVELTGHSIFDFTHPCDHEEIRENLSLKNGSGFGKKSKDMSTERDFFMRMKCTVTNRGRTVNLKSATWKVLHCTGQVKVYNNCPPHNSLCGYKEPLLSCLIIMCEPIQHPSHMDIPLDSKTFLSRHSMDMKFTYCDDRITELIGYHPEELLGRSAYEFYHALDSENMTKSHQNLCTKGQVVSGQYRMLAKHGGYVWLETQGTVIYNPRNLQPQCIMCVNYVLSEIEKNDVVFSMDQTESLFKPHLMAMNSIFDSSGKGAVSEKSNFLFTKLKEEPEELAQLAPTPGDAIISLDFGNQNFEESSAYGKAILPPSQPWATELRSHSTQSEAGSLPAFTVPQAAAPGSTTPSATSSSSSCSTPNSPEDYYTSLDNDLKIEVIEKLFAMDTEAKDOCSTQTDFNELDLETLAPYIPMDGEDFQLSPICPEERLLAENPQSTPQHCFSAMTNIFQPLAPVAPHSPFLLDKFQQQLESKKTEPEHRPMSSIFFDAGSKASLPPCCGQASTPLSSMGGRSNTQWPPDPPLHFGPTKWAVGDQRTEFLGAAPLGPPVSPPHVSTFKTRSAKGFGARGPDVLSPAMVALSNKLKLKRQLEYEEQAFQDLSGGDPPGGSTSHLMWKRMKNLRGGSCPLMPDKPLSANVPNDKFTQNPMRGLGHPLRHLPLPQPPSAISPGENSKSRFPPQCYATQYQDYSLSSAHKVSGMASRLLGPSFESYLLPELTRYDCEVNVPVLGSSTLLQGGDLLRALDQAT

[0037] The N-terminal bHLH domain (which plays a role in DNA binding)and the C-terminal transactivation domain are highlighted (in bold andunderlined type, respectively).

[0038] Table 5 shows the nucleotide sequence DNA encoding human wildtype EPAS1. Nucleotides encoding the first amino acid of EPAS1 areunderlined. TABLE 5 Nucleotide sequence of human EPAS1 cDNA 1 cctgactgcgcggggcgctc gggacctgcg cgcacctcgg accttcacca cccgcccggg (SEQ ID NO:7) 61ccgcggggag cggacgaggg ccacagcccc ccacccgcca gggagcccag gtgctcggcg 121tctgaacgtc tcaaagggcc acagcgacaa tgacagctga caaggagaag aaaaggagta 181gctcggagag gaggaaggag aagtcccggg atgctgcgcg gtgccggcgg agcaaggaga 241cggaggtgtt ctatgagctg gcccatgagc cgcctctgcc ccacagtgtg agctcccatc 301tggacaaggc ctccatcatg cgactggaaa tcagcttcct gcgaacacac aagctcctct 361cctcagtttg ctctgaaaac gagtccgaag ccgaagctga ccagcagatg gacaacttgt 421acctgaaagc cttggagggt ttcattgccg tggtgaccca agatggcgac atgatctttc 481tgtcagaaaa catcagcaag ttcatgggac ttacacaggt ggagctaaca ggacatagta 541tctttgactt cactcatccc tgcgaccatg aggagattcg tgagaacctg agtctcaaaa 601atggctctgg ttttgggaaa aaaagcaaag acatgtccac agagcgggac ttcttcatga 661ggatgaagtg cacggtcacc aacagaggcc gtactgtcaa cctcaagtca gccacctgga 721aggtcttgca ctgcacgggc caggtgaaag tctacaacaa ctgccctcct cacaatagtc 781tgtgtggcta caaggagccc ctgctgtcct gcctcatcat catgtgtgaa ccaatccagc 841acccatccca catggacatc cccctggata gcaagacctt cctgagccgc cacagcatgg 901acatgaagtt cacctactgt gatgacagaa tcacagaact gattggttac caccctgagg 961agctgcttgg ccgctcagcc tatgaattct accatgcgct agactccgag aacatgacca 1021agagtcacca gaacctgtgc accaagggtc aggtagtaag tggccagtac cggatgctcg 1081caaagcatgg gggctacgtg tggctggaga cccaggggac ggtcatctac aaccctcgca 1141acctgcagcc ccagtgcatc atgtgtgtca actacgtcct gagtgagatt gagaagaatg 1201acgtggtgtt ctccatggac cagactgaat ccctgttcaa gccccacctg atggccatga 1261acagcatctt tgatagcagt ggcaaggggg ctgtgtctga gaagagtaac ttcctattca 1321ccaagctaaa ggaggagccc gaggagctgg cccagctggc tcccacccca ggagacgcca 1381tcatctctct ggatttcggg aatcagaact tcgaggagtc ctcagcctat ggcaaggcca 1441tcctgccccc gagccagcca tgggccacgg agttgaggag ccacagcacc cagagcgagg 1501ctgggagcct gcctgccttc accgtgcccc aggcagctgc cccgggcagc accaccccca 1561gtgccaccag cagcagcagc agctgctcca cgcccaatag ccctgaagac tattacacat 1621ctttggacaa cgacctgaag attgaagtga ttgagaagct cttcgccatg gacacagagg 1681ccaaggacca atgcagtacc cagacggatt tcaatgagct ggacttggag acactggcac 1741cctatatccc catggacggg gaagacttcc agctaagccc catctgcccc gaggagcggc 1801tcttggcgga gaacccacag tccacccccc agcactgctt cagtgccatg acaaacatct 1861tccagccact ggcccctgta gccccgcaca gtcccttcct cctggacaag tttcagcagc 1921agctggagag caagaagaca gagcccgagc accggcccat gtcctccatc ttctttgatg 1981ccggaagcaa agcatccctg ccaccgtgct gtggccaggc cagcacccct ctctcttcca 2041tggggggcag atccaatacc cagtggcccc cagatccacc attacatttt gggcccacaa 2101agtgggccgt cggggatcag cgcacagagt tcttgggagc agcgccgttg gggccccctg 2161tctctccacc ccatgtctcc accttcaaga caaggtctgc aaagggtttt ggggctcgag 2221gcccagacgc gctgagtccg gccatggtag ccctctccaa caagctgaag ctgaagcgac 2281agctggagta tgaagagcaa gccttccagg acctgagcgg gggggaccca cctggtggca 2341gcacctcaca tttgatgtgg aaacggatga agaacctcag gggtgggagc tgccctttga 2401tgccggacaa gccactgagc gcaaatgtac ccaatgataa gttcacccaa aaccccatga 2461ggggcctggg ccatcccctg agacatctgc cgctgccaca gcctccatct gccatcagtc 2521ccggggagaa cagcaagagc aggttccccc cacagtgcta cgccacccag taccaggact 2581acagcctgtc gtcagcccac aaggtgtcag gcatggcaag ccggctgctc gggccctcat 2641ttgagtccta cctgctgccc gaactgacca gatatgactg tgaggtgaac gtgcccgtgc 2701tgggaagctc cacgctcctg caaggagggg acctcctcag agccctggac caggccacct 2761gagccaggcc ttctacctgg gcagcacctc tgccgacgcc gtcccaccag cttcaccc

[0039] Hypoxia inducible factor-1α (HIF-1α) is another member of the PASfamily to which EPAS1 belongs. Transcription factors of this family usethe bHLH and PAS domains to form heterodimers that subsequently bind totarget genes and regulate important biological processes.

[0040] EPAS1 plays a role in the regulation of angiogenic factors suchas VEGF, VEGF-R such as KDR/flk-1 and flt-1, and Tie2. EPAS1, a nuclearprotein with a basic helix-loop-helix (bHLH)/PAS domain, is expressedpreferentially in endothelial cells. EPAS1 transcription factor or DNAencoding all or part of EPAS1 (e.g., a fragment containing theC-terminal activation domain) is administered to individuals to promoteangiogenesis. To inhibit angiogenesis, EPAS1 antisense sequences areadministered to cells to decrease intracellular production of EPAS1 geneproduct. Administration of DNA encoding an EPAS1-specific antibody(e.g., EPAS1 intrabodies) or EPAS1 dominant negative mutants can also beadministered cells to inhibit EPAS1 function, e.g., by inhibitingbinding of EPAS1 to cis-acting regulatory sequences of VEGF, VEGF-R, orTie2 genes or by inhibiting EPAS1 transactivation of gene transcription.By regulating transcription of VEGF, VEGF-Rs, and Tie2, EPAS1 is usefulto modulate vasculogenesis and angiogenesis.

Production of ARNT4-Specific Antibodies

[0041] Anti-ARNT4 antibodies are obtained by techniques well known inthe art. Such antibodies can be polyclonal or monoclonal. Polyclonalantibodies are obtained, for example, by the methods described in Ghoseet al., Methods in Enzymology, Vol. 93, 326-327, 1983. For example, aARNT4 polypeptide, or an antigenic fragment thereof, can be used as animmunogen to stimulate the production of ARNT4-reactive polyclonalantibodies in the antisera of animals such as rabbits, goats, sheep, orrodents. Antigenic polypeptides useful as immunogens includepolypeptides which contain a bHLH domain/PAS domain.

[0042] Monoclonal antibodies are obtained by standard techniques such asthose described by Milstein and Kohler in Nature, 256:495-97, 1975, oras modified by Gerhard, Monoclonal Antibodies, Plenum Press, 1980, pages370-371. Hybridomas are screened to identify those producing antibodiesthat are highly specific for an ARNT4 polypeptide. Preferably, theantibody will have an affinity of at least about 108 liters/mole andmore preferably, an affinity of at least about 109 liters/mole.

ARNT4-Deficient Mice

[0043] To further investigate the role of ARNT4 in vivo, ARNT4 knockoutmice (ARNT4-deficient mice) are generated by homologous recombination. Agene targeting construct for generating ARNT4-deficient mice is madeusing a targeted gene deletion strategy using standard methods. Thedeletion in the ARNT4 gene renders the ARNT4 polypeptide non-functional.The linearized targeting construct is transfected into murine D3embryonic stem (ES) cells, and a clone with the correct homologousrecombination (yielding the appropriately disrupted ARNT4 gene) isinjected into blastocysts and used to generate ARNT4-deficient mice.

Activation of the KDR/flk-1 Promoter by EPAS1

[0044] EPAS1 and KDR/flk-1 transcripts were found to colocalize invascular endothelial cells in mouse embryonic and adult tissue. To studythe expression of EPAS1 relative to KDR/flk-1, a plasmid containing 4.0kb of human KDR/flk-1 5′-flanking sequence linked to the luciferasereporter gene and a second vector containing DNA encoding either EPAS1or another bHLH-PAS domain transcription factor HIF-1α werecotransfected into bovine aortic endothelial cells (BAEC). EPAS1 but notHIF-1α markedly increased KDR/flk-1 promoter activity in adose-dependent manner, and this induction of the KDR/flk-1 promoter byEPAS1 occurred preferentially in endothelial cells. In contrast, bothEPAS1 and HIF-1α activated the VEGF promoter in a non-endothelialcell-specific manner. This is the first demonstration of transactivationof the KDR/flk-1 promoter by EPAS1. By regulating transcription ofKDR/flk-1 and VEGF, EPAS1 plays an important role in regulatingvasculogenesis and angiogenesis.

Cell Culture

[0045] BAEC were isolated and cultured in DME supplemented with 10% FCS(HyClone, Logan, Utah) and antibiotics according to known procedures.BAEC were passed every 3-5 days, and cells from passages 5-7 were usedfor the transfection experiments. The following cell lines were obtainedfrom the American Type Culture Collection (ATCC) and were cultured inthe same medium as BAEC: HeLa cells (human epidermoid carcinoma cells;ATCC #CRL7396 and NIH 3T3 cells (mouse fibroblasts; ATCC #CRL1888).

RNA Isolation and Northern Analysis

[0046] Total RNA was isolated from mouse organs by guanidiniumisothiocyanate extraction and centrifugation through cesium chlorideaccording to standard protocols. Total RNA (10 μg) was fractionated on a1.3% formaldehyde-agarose gel and transferred to Nitropure filters (MSI,Westborough, Mass.). The filters were then hybridized with ³²P-labeled,randomly primed cDNA probes for 1 h at 68° C. in Quick-hyb solution(Stratagene, La Jolla, Calif.). The hybridized filters were washed in 30mM NaCl, 3 mM sodium citrate, and 0.1% sodium dodecyl sulfate at 55° C.and autoradiographed for 20 h on Kodak XAR film at −80° C. To correctfor differences in RNA loading, the filters were rehybridized with aradiolabeled ribosomal 18S-specific oligonucleotide. A 1.8 kb AccI-AccIfragment of mouse EPAS1 (GENBANK Accession # U81983) was used as aprobe. The 667 (382-1086) bp mouse KDR/flk-1 cDNA fragment was amplifiedby the reverse transcriptase PCR by using mouse lung total RNA. Theforward (5′ GAACTTGGATGCTCTTTGGAAA 3′; SEQ ID NO: 8) and reverse (5′CACTTGCTGGCATCATAAGGC 3′; SEQ ID NO: 9) primers were used to generatePCR fragments that were subcloned into to a pCR 2.1 vector (Invitrogen,Carlsbad, Calif.). Nucleotide sequence authenticity was confirmed by thedideoxy chain termination method.

In Situ Hybridization

[0047] To generate probes for in situ hybridization, a 316 (771-1086) bpmouse EPAS1 CDNA and a 342 (2346-2687) bp mouse KDR/flk-1 cDNA frommouse lung total RNA was amplified by reverse transcriptase PCR with thefollowing primers: EPAS1, forward 5° CATCATGTGTGAGCCAATCCA 3′ (SEQ IDNO: 10) and reverse 5′ GTTGTAGATGACCGTCCCCTG 3′ (SEQ ID NO: 11)KDR/flk-1, forward 5′ TGTACTGAGAGATGGGAACCG 3′ (SEQ ID NO: 12) andreverse 5′ CACTTGCTGGCATCATAAGGC 3′ (SEQ ID NO: 13). PCR fragments weresubcloned into the pCR 2.1 vector in both orientations and theauthenticity of the sequences was confirmed.

[0048] Slides of E9 mouse sections were purchased from Novagen (Madison,Wis.). E12 mice and various adult mouse organs were fixed in 4%paraformaldehyde, dehydrated, and embedded in paraffin. Tissue sections(6 μm thick) were hybridized with a ³⁵S-UTP-labeled antisense cRNA probesynthesized with the T7 RNA polymerase from linearized plasmidscontaining appropriate cDNA fragments using standard techniques. As anegative control, tissue sections were also hybridized with³⁵S-UTP-labeled sense cRNA probes. After hybridization the tissuesections were washed, and the dried tissue sections were then dippedinto Kodak NTB2 emulsion (Eastman Kodak) and exposed for 10-15 days at4° C. The sections were counterstained with hematoxylin and eosin.

Construction of Plasmids

[0049] pGL2-Basic and pGL2-Control contained the firefly luciferasereporter gene (Promega, Madison, Wis.). pGL2-Basic had no promoter,whereas pGL2-Control contained the SV40 promoter and enhancer. ThepGL2-4kb+296 reporter plasmid was constructed by inserting the humanKDR/flk-1 promoter from -4kb to +296 into pGL2-Basic. pVR47/CAT, whichcontains the human VEGF promoter from −2362 to +61 and thechloramphenicol acetyltransferase (CAT) reporter gene sequence, was alsoconstructed using standard techniques.

[0050] The plasmid phEP-lAS was made by cloning the antisense EPAS1 cDNAinto pcDNA3. phEP-1ΔCT, containing a C-terminal deletion mutant of theEPAS1 cDNA, was generated by subcloning a BamHI-XhoI restrictionfragment encoding human EPAS1 amino acids 1-690 into pcDNA3. To generatephHIF-1α, a 2622 bp cDNA fragment containing the entire open readingframe of human HIF-1a was amplified using human leukocyte total RNA andpfu DNA polymerase (Stratagene, La Jolla, Calif.). The sequences of theforward (5′ GTGAAGACATCGCGGGGACC 3′; SEQ ID NO: 14) and reverse (5′GTTTGTGCAGTATTGTAGCCAGG 3′; SEQ ID NO: 15) primers were based on humanHIF-1a cDNA (Wang et al., 1995, Proc. Natl. Acad. Sci. USA.92:5510-5514). The PCR fragment was then cloned into pcDNA3, and thesequence was confirmed. Expression of phEP-1, phEP-1ΔCT, and phHIF-1awas confirmed by in vitro transcription and translation in theTNT-coupled reticulocyte lysate system (Promega, Madison, Wis.)according to the manufacturer's instructions.

Transient Transfection Assays

[0051] Cells were transfected with 1 μg of reporter construct and 6 μgof expression construct by the standard calcium phosphate method. Tocorrect for variability in transfection efficiency againstβ-galactosidase, 1 βg of pCMV-βGAL was cotransfected in all experiments.Cell extracts were prepared 48 h after transfection by a detergent lysismethod (Promega, Madison, Wis.). Luciferase activity was measured induplicate for all samples with an EC&G Autolumat 953 (Gaithersburg, Md.)luminometer by the Promega luciferase assay. CAT activity was assayed bya two-phase fluor diffusion method. β-galactosidase activity was assayedusing standard methods. The ratio of luciferase or CAT activity toβ-galactosidase activity in each sample served as a measure ofnormalized luciferase or CAT activity. Each construct was transfected atleast four times, and each transfection was done in triplicate. Data foreach construct are presented as the mean ± SE.

Statistics

[0052] Comparisons between groups were made by a factorial analysis ofvariance followed by Fisher's least significant difference test whenappropriate. Statistical significance was accepted at p<0.05.

[0053] Tissue distribution of EPAS1 and KDR/flk-1 in adult mice Northernblot analysis was performed with RNA prepared from various adult mousetissues. EPAS1 mRNA was abundant in the lung, heart, and aorta, organsknown to be rich in vascular endothelial cells. When the same blot washybridized to a mouse KDR/flk-1 probe, the expression pattern ofKDR/flk-1 was identical to that of EPAS1. In situ hybridization wasperformed using an antisense mouse EPAS1 probe to determine which cellsin the aorta expressed EPAS1. The EPAS1 message localized to the luminallayer, and the antisense EPAS1 probe but not the sense EPAS1 probehybridized to the endothelial cells of the aorta.

Tissue Distribution of EPAS1 and KDR/flk-1 in Developing Mouse Embryos

[0054] To characterize the temporal and spatial patterns of EPAS1 andKDR/flk-1 expression in developing mouse embryos, in situ hybridizationwas performed with EPAS1 and KDR/flk-1 probes. In embryonic-day (E)9mice, EPAS1 mRNA was visible in the dorsal aorta, the endocardium of thedeveloping outflow tract, the ventricle, and the perineural vascularplexus. KDR/flk-1 mRNA was expressed similarly in the same organs. Atthe E9 stage of development, the mouse aorta is composed mainly of asingle layer of endothelial cells. Both EPAS1 and KDR/flk-1 wereexpressed in endothelial cells of the aorta and other organs. At E12.5,EPAS1 mRNA was visible in the intervertebral blood vessels, heart,vascular plexuses in the meninges surrounding both the spinal cord andthe brain, and choroid plexus. The distribution of KDR/flk-1 MRNA atE12.5 was strikingly similar. The EPAS1 and KDR/flk-1 mRNAs were bothdetected in endothelial cells of the blood vessels at highermagnification as well.

Transactivation of the KDR/flk-1 Promoter by EPAS1 in a Dose-DependentManner

[0055] The colocalization of EPAS1 and KDR/flk-1 indicates that EPAS1 isimportant in regulating KDR/flk-1 expression. To test the role of EPAS1in regulation of protein expression, a human EPAS1 expression plasmid(phEP-1) and a reporter plasmid (pGL2-4kb+296) containing approximately4.0 kb of the human KDR/flk-1 5′-flanking sequence linked to aluciferase reporter gene were cotransfected into BAEC. EPAS1 increasedKDR/flk-1 promoter activity in a dose-dependent manner (FIG. 1A). Aslittle as 2 μg of EPAS1 expression vector phEP-1 increased the promoteractivity of KDR/flk-1 by 3-fold, and 6 μg of the EPAS1 vector increasedluciferase activity by 12.9-fold. Upregulation of KDR/flk-1 promoteractivity by EPAS1 was specific, since cotransfection of the EPAS1expression vector had no effect on the activity of pGL2-Control vectordriven by the potent SV40 promoter and enhancer.

[0056] To identify the EPAS1 domain which participates intransactivation of the KDR/flk-1 promoter, plasmid phEP-lACT wasconstructed to express a truncated form of EPAS1 lacking its 180C-terminal amino acids. Deletion of the 180 C-terminal amino acids ofEPAS1 completely abolished its ability to transactivate the KDR/flk-1promoter (FIG. 1B). These data indicate that the 180 C-terminal aminoacids of EPAS1 are necessary for transactivation of the KDR/flk-1promoter.

[0057] These data indicate that induction of the mRNA for KDR/flk-1colocalizes with that of the mRNA for EPAS1 in vascular endothelialcells from fetal as well as adult mice. EPAS1 also transactivates thepromoter of Tie2, which, like KDR/flk-1, is an endothelial cell-specifictyrosine kinase. Expression of Tie2 in endothelial cells is high duringfetal development but barely detectable in adulthood. In contrast,expression of EPAS1 in endothelial cells is high in fetuses as well asadults. Thus, the target gene for EPAS1 in adults is a VEGF-R such asKDR/flk-1 or flt-1 (as well as VEGF) as evidenced by the data showingthat EPAS1 markedly induces KDR/flk-1 promoter activity.

EPAS1 but not HIF-1αTransactivates the KDR/flk-1 Promoter Preferentiallyin Vascular Endothelial Cells

[0058] To determine whether another member of the bHLH/PAS familytransactivated the KDR/flk-1 promoter, the EPAS1 or HIF-1α expressionplasmid and the KDR/flk-1 plasmid pGL2-4kb+296 were cotransfected intoBAEC, HeLa cells, and NIH 3T3 cells. EPAS1 expression plasmids in thesense (phEP-1) but not the antisense (phEP-1AS) orientation activatedthe KDR/flk-1 promoter (FIG. 2A), indicating that the transactivatingeffect is cell-specific. Although the EPAS1 plasmid markedly increasedKDR/flk-1 promoter activity in vascular endothelial cells, it had littleeffect on KDR/flk-1 promoter activity in HeLa or NIH 3T3 cells (FIG.2A). HIF-1α had no effect on KDR/flk-1 promoter activity in all threecell types. The EPAS1 or HIF-1α expression plasmid was thencotransfected with a reporter plasmid containing the VEGF promoter,pVR47/CAT, to determine whether the differential effects of EPAS1 andHIF-1a were unique to the KDR/flk-1 promoter. In contrast to itscell-specific effect on the KDR/flk-1 promoter (FIG. 2A), EPAS1transactivated the VEGF promoter in all three cell types (FIG. 2B).Induction was highest in HeLa cells. Furthermore, HIF-1a increased VEGFpromoter activity in BAEC and HeLa cells (FIG. 2B). These data indicatethat the transactivating effect of EPAS1 depends on both the promoterand the cell type.

[0059] Although EPAS1 transactivated the KDR/flk promoter preferentiallyin endothelial cells (FIG. 2A), it activated the VEGF promoter in anon-endothelial cell-specific manner (FIG. 2B). Despite the fact thatHIF-1α is 48% homologous to EPAS1, HIF-1α had no effect on the KDR/flk-1promoter. In contrast, HIF-1α transactivated the VEGF promoter. Thus,the effect of EPAS1 on the KDR/flk-1 promoter is specific and cannot bereplaced by other members of the PAS family of transcription factors.

[0060] EPAS1 heterodimerizes with the aryl hydrocarbon receptor nucleartranslocator and transactivates the promoter of Tie2. EPAS1 alsomarkedly increases the promoter activity of KDR/flk-1 and VEGF. Micedeficient in the aryl hydrocarbon receptor nuclear translocator are notviable past E10.5, and the yolk sac shows defective angiogenesis. Thesedata indicate that EPAS1 functions as a nodal transcription factor byregulating expression of VEGF, KDR/flk-1, and Tie2 during vasculogenesisand angiogenesis.

Characterization of Functional Domains of EPAS1

[0061] Functional domains of EPAS1 were identified as follows. The geneencoding VEGF has a cis-acting regulatory sequence to which EPAS1 binds(GCCCTACGTGCTGTCTCA; SEQ ID NO:1) in its 5′ flanking region. Incotransfection experiments in BAEC, the EPAS1 expression plasmidactivated by 30-fold a CAT reporter plasmid containing 2.3 kb of VEGF 5′flanking sequence (containing SEQ ID NO: 1) but not a similar plasmiddiffering only by a mutation in an amino acid of SEQ ID NO: 1. Thesedata indicate that EPAS1 activates the VEGF promoter by binding to DNAcontaining the sequence of SEQ ID NO: 1. To further characterize domainsof EPAS1 which function to activate promoters of angiogenic factors inendothelial cells, e.g., the VEGF promoter or VEGF-R promoters, BAECwere cotransfected with expression plasmids encoding EPAS1 mutants andthe reporter plasmid. Eight mutants were tested. Deletion of the basicregion (bHLH region) of EPAS1 (SEQ ID NO: 3) completely abolished itsability to induce transcription from the VEGF promoter, indicating thatbinding of EPAS1 to the cis-acting element though this basic region iscritical. Deletion of 180 amino acids from the C-terminus of EPAS1 haslittle or no effect on the transcriptional transactivation activity ofEPAS1 for the VEGF promoter; however, a deletion of the C-terminal 385amino acids abolished the ability of EPAS1 to activate the VEGFpromoter, indicating the presence of a transactivation domain in theportion of EPAS1 spanning amino acids 486-690. Further fine deletionanalyses indicated that the transactivation domain of EPAS1 spans aminoacids 486-639. An EPAS1 mutant polypeptide lacking the amino acidsequence of SEQ ID NO: 2, e.g., an EPAS1 with the amino acid sequence ofSEQ ID NO: 4, functions as a dominant negative mutant EPAS1 because itinhibited transactivation of the VEGF promoter by wild type EPAS1 in adose-dependent manner. Deletion analysis is also used to identifydomains of EPAS1 which participate in heterodimer formation with ARNT4.

[0062] To characterize domains of ARNT4 which function to heterodimerizewith EPAS1 and activate promoters of angiogenic factors in endothelialcells, e.g., the VEGF promoter or VEGF-R promoters, BAEC arecotransfected with expression plasmids encoding EPASI and ARNT4 deletionmutants and the reporter plasmid as described above. Domains of ARNT4which participate in EPAS1 heterodimer formation with EPAS1 areidentified using the yeast two-hybrid assay or a gel mobility assay. Forexample, those mutants which fail to activate the VEGF/luciferasepromoter cannot form functional dimers with EPAS1.

[0063] This assay is also used to identify compounds which inhibit ordecrease formation of functional ARNT4/EPAS1 heterodimers, and thus,inhibit angiogenesis. In such an assay, expression plasmids which encodewild type or functional fragments of ARNT4 and EPAS1 are cotransfectedwith a VEGF/luciferase reporter plasmid into an endothelial cell in thepresence and absence of a candidate compound. A decrease in the amountof transactivation of the VEGF promoter (e.g., as measured by a standardluciferase assay) in the presence of the compound compared to the amountin the absence of the candidate compound indicates that the compoundinhibits angiogenesis (by inhibiting ARNT4/EPAS1 transactivation of theVEGF promoter).

Generation of a Dominant-Negative EPAS1 Mutants

[0064] An adenoviral construct which expresses EPAS1 was generated.Overexpression of EPAS1 dramatically induced VEGF mRNA in humanumbilical endothelial cells. In cotransfection experiments, EPAS1transactivated the VEGF promoter via the HIF-1 binding site. Thistransactivation was further enhanced by hypoxia. Cotransfection of anaryl hydrocarbon receptor nuclear translocator (ARNT) expression plasmidand EPAS1 expression plasmid synergistically transactivated the VEGFpromoter, indicating that heterodimerization of EPAS1 and ARNT iscrucial for the transactivation of the VEGF promoter (FIG. 5). Using agel shift analysis, EPAS1 (but not HIF-1) formed dimers with ARNT4 andbound to the HIF-1 binding site of the VEGF promoter.

[0065] Deletion analysis of EPAS1 further defined a potenttransactivation domain to span amino acids 486-639 of human EPAS1 (SEQID NO: 6). The transactivation domain is essential for EPAS1 totransactivate the VEGF promoter. The ability of this domain to activatetranscription was confirmed using the GAL4 fusion protein system.Finally, a truncated EPAS1 lacking the transactivation domain (e.g., anEPAS1 polypeptide lacking amino acids 486-690 of SEQ ID NO: 6 or anEPASI polypeptide lacking amino acids 486-639 of SEQ ID NO: 6) retainedits ability to form heterodimers and to bind the HIF-1 binding site.These data indicate that the mutated EPAS1 polypeptides with lack aminoacids in the transactivation domain are dominant negative mutantsbecause they sequester ARNT and prevent the formation of functionalEPAS1/ARNT and HIF-1α/ARNT heterodimers. For example, the EPAS1polypeptide which lacked amino acids 486-639 of SEQ ID NO: 6 potentlyinhibited the induction of the VEGF promoter by EPAS1 and HIF-1α.Transfection of endothelial cells with an adenovirus construct encodingthis mutant inhibited VEGF MRNA induction by hypoxia. These resultsindicate that EPAS1 is an important regulator of VEGF gene expressionand that dominant negative EPAS1 mutants (e.g., EPAS1 polypeptideslacking all or part of the transactivation domain (SEQ ID NO: 2))inhibit VEGF promoter activity, and in turn, VEGF expression andangiogenesis.

Identification of Compounds which Modulate EPAS1 Binding toCis-Regulatory Sequences

[0066] Modulation of the angiogenesis is achieved by contacting thevascular cells such as vascular endothelial cells with a compound thatblocks or enhances EPAS1 binding to cis-acting regulatory sequences ofVEGF, VEGF-Rs, or other angiogenic factors in endothelial cells such asTie2. Such a compound can be identified by methods ranging from rationaldrug design to screening of random compounds. The latter method ispreferable, as simple and rapid assays for testing such compounds areavailable. oligonucleotides and small organic molecules are desirablecandidate compounds for this analysis.

[0067] The screening of compounds for the ability to modulateangiogenesis by affecting EPAS1 transactivation of transcription ofangiogenic factors may be carried out using in vitro biochemical assays,cell culture assays, or animal model systems. For example, in abiochemical assay, labeled EPAS1 (e.g., EPAS1 labeled with afluorochrome or a radioisotope) is applied to a column containingimmobilized DNA containing the cis-acting regulatory sequence.Alternatively, ARNT4 is immobilized on the column. In this manner,compounds which inhibit ARNT4/EPAS1 heterodimerization may beidentified. A candidate compound is applied to the column before, after,or simultaneously with the labeled EPAS1, and the amount of labeledprotein bound to the column in the presence of the compound isdetermined by conventional methods. A compound tests positive forinhibiting EPAS1 binding (thereby having the effect of inhibitingangiogenesis) if the amount of labeled protein bound in the presence ofthe compound is lower than the amount bound in its absence. Conversely,a compound tests positive for enhancing EPAS1 binding (thereby havingthe effect of enhancing angiogenesis) if the amount of labeled proteinbound in the presence of the compound is greater than the amount boundin its absence. In a variation of the above-described biochemical assay,binding of labeled DNA to immobilized EPAS1 is measured.

[0068] As mentioned above, candidate compounds may also be screenedusing cell culture assays. Cells expressing EPAS1, either naturally orafter introduction into the cells of genes encoding EPAS1 are culturedin the presence of the candidate compound. The level of EPAS1 binding inthe cell may be inferred using any of several assays. For example,levels of expression of EPAS1 regulated genes (e.g., genes encodingVEGF, VEGF-Rs such as KDR/flk-1 or flt-1) in the cell may determinedusing, e.g., Northern blot analysis, RNAse protection analysis,immunohistochemistry, or other standard methods. Compounds identified ashaving the desired effect, either enhancing or inhibiting EPAS1 binding,can be tested further in appropriate animal models, e.g., an animal witha tumor or atherosclerotic lesion.

[0069] Compounds found to inhibit EPAS1 binding to cis-acting regulatorysequences of geres encoding angiogenic factors may be used in methodsfor nhibiting pathogenic angiogenesis in order to, e.g., prevent ortreat tumor progression or the progression of an atherosclerotic lesion.Compounds found to enhance EPAS1 binding may be used in methods totherapeutically promote new blood vessel formation in adult mammals asdiscussed above.

[0070] The therapeutic compounds identified using the methods of theinvention may be administered to a patient by any appropriate method forthe particular compound, e.g., orally, intravenously, parenterally,transdermally, transmucosally, by inhalation, or by surgery orimplantation at or near the site where the effect of the compound isdesired (e.g., with the compound being in the form of a solid orsemi-solid biologically compatible and resorbable matrix). Therapeuticdoses are determined specifically for each compound, most beingadministered within the range of 0.001 to 100.0 mg/kg body weight, orwithin a range that is clinically determined to be appropriate by oneskilled in the art.

Identification and Molecular Cloning of the EPAS1 Binding Partner,ARNT-4

[0071] Compositions which interact with EPAS1 were identified byscreening for endothelial cell proteins which bind to EPAS1. Yeast twohybrid screening of a human umbilical endothelial cell cDNA library wascarried out using EPAS1 as a bait. One of the clones isolated encoded anovel bHLH/PAS protein which was found to have similarity witharylhydrocarbon nuclear translocator 3 (Arnt3), a member of bHLH/PASprotein which heterodimerizes with Clock, a gene product involved inregulation of mammalian circadian rhythm. The isolated clone was namedARNT4. As described above, the CLUSTAL W sequence alignment system wasused to compare the sequences of ARNT4 with the most closely relatedknown DNA and/or amino acid sequences. With respect to DNA (comparisonof coding sequences; untranslated regions excluded), the sequences ofhARNT and hARNT4 were found to be 35% identical; the sequences of hBMALlb and hARNT4 were found to be 56% identical; and the sequences of hARNTand hBMAL lb were found to be 37% identical. Nucleotide sequencecomparisons using the CLUSTAL W system were carried out using thefollowing parameters: KTUP=2; gap penalty=5; top diagonals=4; and windowsize=4. With respect to the proteins, the amino acid sequences of hARNTand hARNT4 were found to be 23% identical; the sequences of hBMAL lb andhARNT4 were found to be 49% identical; and the sequences of hARNT andhBMAL lb were found to be 26% identical. Amino acid sequence comparisonsusing the CLUSTAL W system were carried out using the followingparameters: KTUP=1; gap penalty=3; top diagonals=5; and window size=5.

[0072] Northern analysis of human tissue revealed that this gene ishighly expressed in brain, heart and placenta. In the brain, expressionwas high in the thalamus and amygdala, an almond-shaped component of thelimbic system located in the temporal lobe of the brain.

[0073] Expression within human cultured cells demonstrated highest mRNAlevels in vascular endothelial cells and smooth muscle cells. ARNT4 wasshown to interact with EPASI using the yeast two-hybrid assay (FIG. 4).In a gel mobility shift assay using hypoxia responsive element of VEGFgene as the probe, ARNT4 formed a heterodimer with EPAS1 and bound tothe hypoxia responsive element of the VEGF gene.

[0074] An expression plasmid encoding EPAS1 and an expression plasmidencoding ARNT4 were cotransfected with a VEGF/luciferase reporterplasmid into bovine aortic endothelial cells. Coexpression of ARNT4 andEPAS1 markedly transactivated the VEGF promoter (FIG. 5), and thistransactivation was further enhanced by hypoxia. These data indicatethat the heterodimer EPAS1/ARNT4 is activated under hypoxic conditions.Taken together, these results indicate that ARNT4, a novel bHLH/PASprotein, is an important regulator of VEGF gene expression especially invascular system. TABLE 6 Human ARNT4 amino acid sequence M A A E E 5(SEQ ID NO:19) 6 E A A A G G K V L R E E N Q C I A P V V 25 26 S S R V SP G T R P T A M G S F S S H M 45 46 T E F P R K R K G S D S D P S Q V ED G 65 66 E H Q V K M K A F R E A H S Q T E K R R 85 86 R D K M N N L IE E L S A M I P Q C N P 105 106 M A R K L D K L T V L R M A V Q H L R S125 126 L K G L T N S Y V G S N Y R P S F L Q D 145 146 N E L R H L I LK T A E G F L F V V G C 165 166 E R G K I L F V S K S V S K I L N Y D Q185 186 A S L T G Q S L F D F L H P K D V A K V 205 206 K E Q L S S F DI S P R E K L I D A K T 225 226 G L Q V H S N L H A G R T R V Y S G S R245 246 R S F F C R I K S C K I S V K E E H G C 265 266 L P N S K K K EH R K F Y T I H C T G Y 285 286 L R S W P P N I V G M E E E R N S K K D305 306 N S N F T C L V A I G R L Q P Y I V P Q 325 326 N S G E I N V KP T E F I T R F A V N G 345 346 K F V Y V D Q R A T A I L G Y L P Q E L365 366 L G T S C Y E Y F H Q D D H N N L T D K 385 386 H K A V L Q S KE K I L T D S Y K F R A 405 406 K D G S F V T L K S Q W F S F T N P W T425 426 K E L E Y I V S V N T L V L G H S E P G 445 446 E A S F L P C SS Q S S E E S S R Q S C 465 466 M S V P G M S T G T V L G A G S I G T D485 486 I A N E I L D L Q R L Q S S S Y L D D S 505 506 S P T G L M K DT H T V N C R S M S N K 525 526 E L F P P S P S E M G E L E A T R Q N Q545 546 S T V A V H S H E P L L S D G A Q L D F 565 566 D A L C D N D DT A M A A F M N Y L E A 585 586 E G G L G D P G D F S D I Q W T L 602

[0075] TABLE 7 Human ARNT4 cDNActccagtccgcatgctcagtagctgctgccggccgggctgcggggcggcgtccgctgcgc (SEQ IDNO:20) gcctacgggctgcggtggcggccgccgcggcacccggcagggcccgccagtccccgcttccctgctccagagccgccgcctgggccggggcagggcgggcccggggctcctccatgctgccagccgccgggctgcggagccgaccaagtggctcctgcg ATG GCG GCG GAA GAG GAG GCT GCGGCG GGA GGT AAA GTG TTG AGA GAG GAG AAC CAG TGC ATT GCT CCT GTG GTT TCCAGC CGC GTG AGT CCA GGG ACA AGA CCA ACA GCT ATG GGG TCT TTC AGC TCA CACATG ACA GAG TTT CCA CGA AAA CGC AAA GGA AGT GAT TCA GAC CCA TCC CAA GTGGAA GAT GGT GAA CAC CAA GTT AAA ATG AAG GCC TTC AGA GAA GCT CAT AGC CAAACT GAA AAG CGG AGG AGA GAT AAA ATG AAT AAC CTG ATT GAA GAA CTG TCT GCAATG ATC CCT CAG TGC AAC CCC ATG GCG CGT AAA CTG GAC AAA CTT ACA GTT TTAAGA ATG GCT GTT CAA CAC TTG AGA TCT TTA AAA GGC TTG ACA AAT TCT TAT GTGGGA AGT AAT TAT AGA CCA TCA TTT CTT CAG GAT AAT GAG CTC AGA CAT TTA ATCCTT AAG ACT GCA GAA GGC TTC TTA TTT GTG GTT GGA TGT GAA AGA GGA AAA ATTCTC TTC GTT TCT AAG TCA GTC TCC AAA ATA CTT AAT TAT GAT CAG GCT AGT TTGACT GGA CAA AGC TTA TTT GAC TTC TTA CAT CCA AAA GAT GTT GCC AAA GTA AAGGAA CAA CTT TCT TCT TTT GAT ATT TCA CCA AGA GAA AAG CTA ATA GAT GCC AAAACT GGT TTG CAA GTT CAC AGT AAT CTC CAC GCT GGA AGG ACA CGT GTG TAT TCTGGC TCA AGA CGA TCT TTT TTC TGT CGG ATA AAG AGT TGT AAA ATC TCT GTC AAAGAA GAG CAT GGA TGC TTA CCC AAC TCA AAG AAG AAA GAG CAC AGA AAA TTC TATACT ATC CAT TGC ACT GGT TAC TTG AGA AGC TGG CCT CCA AAT ATT GTT GGA ATGGAA GAA GAA AGG AAC AGT AAG AAA GAC AAC AGT AAT TTT ACC TGC CTT GTG GCCATT GGA AGA TTA CAG CCA TAT ATT GTT CCA CAG AAC AGT GGA GAG ATT AAT GTGAAA CCA ACT GAA TTT ATA ACC CGG TTT GCA GTG AAT GGA AAA TTT GTC TAT GTAGAT CAA AGG GCA ACA GCG ATT TTA GGA TAT CTG CCT CAG GAA CTT TTG GGA ACTTCT TGT TAT GAA TAT TTT CAT CAA GAT GAC CAC AAT AAT TTG ACT GAC AAG CACAAA GCA GTT CTA CAG AGT AAG GAG AAA ATA CTT ACA GAT TCC TAC AAA TTC AGAGCA AAA GAT GGC TCT TTT GTA ACT TTA AAA AGC CAA TGG TTT AGT TTC ACA AATCCT TGG ACA AAA GAA CTG GAA TAT ATT GTA TCT GTC AAC ACT TTA GTT TTG GGACAT AGT GAG CCT GGA GAA GCA TCA TTT TTA CCT TGT AGC TCT CAA TCA TCA GAAGAA TCC TCT AGA CAG TCC TGT ATG AGT GTA CCT GGA ATG TCT ACT GGA ACA GTACTT GGT GCT GGT AGT ATT GGA ACA GAT ATT GCA AAT GAA ATT CTG GAT TTA CAGAGG TTA CAG TCT TCT TCA TAC CTT GAT GAT TCG AGT CCA ACA GGT TTA ATG AAAGAT ACT CAT ACT GTA AAC TGC AGG AGT ATG TCA AAT AAG GAG TTG TTT CCA CCAAGT CCT TCT GAA ATG GGG GAG CTA GAG GCT ACC AGG CAA AAC CAG AGT ACT GTTGCT GTC CAC AGC CAT GAG CCA CTC CTC AGT GAT GGT GCA CAG TTG GAT TTC GATGCC CTA TGT GAC AAT GAT GAC ACA GCC ATG GCT GCA TTT ATG AAT TAC TTA GAAGCA GAG GGG GGC CTG GGA GAC CCT GGG GAC TTC AGT GAC ATC CAG TOG ACC CTCtagcctttgatttttaactccaaaaatgagaaacattttaaagcattatttacgaaaaaactgtctcaactattcttaagtactgtattgatattgtttgtatcttttattaatgttctaccactttttatagatttgcatcttcctgtcacagggatgtggggaaatacgttttcctcccaagagaaccaagtttattatagactcctttattcagtgaaatggcttataatccactagttgccatatttttgctaaaatatttctaaccaagaatactacttacatattgttttggctttgttttatttttgatgcagttttttttagttgaggtaatgtaatatattgatgttttcctttgtgtctaagattgatttataatagtaggtttgtataatttggaacattttccatgccttgcgaatttccttaattgaggatagggcttacacactttaagaaaacagtgagtacttgaacatttaaagggacagtgcaatttatagtcataatcacattgaatactgtatttgatctttggagacttaggcaagcacagagctgggatatttatgctcagttgagcactttaagatgaattttaagtgagatgatttcttgcttaaaactcagaaagtcaaaagagtttcagctttccttacagaaaaggaaggatcttgggccctagatcttggggattaacctctgcatataagatttactcttaataggccagacgtggtgctcacgcctgtaatcccagtactttgggaggctgagacgggcagatcacttgaggtcaggagttcaagaccagcctggccaatatggtgaaaccccgtttctactaaaaatacaaaaaaaattacccaggcactcactcttgaggtaactaaccaactcccacgataatgacagtccattcatgagcgcaaaggcctcatgacctaatggcacacacctgtaatcccaactgcttgggaggctgaggcgagaggattgcttgaacctgggaggcagaggttgcagtgagccgagatcgcaccactgcactccagtctgggcaacagagtgagacttcatctcaaaaaaagtaaaaaaaaagatttaatataatcactgaagatctctattatagatagattaggtttttgacattggaaacatacttagggatagatttgtcctaaaggaaaaaagtaggcccgggcagattaaatgtcttgtgtaaagtcacacattaaattcagtcacacattaaattcatagagttttaaatgtttaatgtatataaaccagtttctttatacacatttgggaaaacattggtctcacagattaaatgattaactaactgacccaggaactagttgtagctttctaagtaattaggcaattacagttattgcctgtaaccaaaggtaataaaacaaaatgacaagtacatgtttaaaattatgaggcaatgagaaataatttaaaaaccaattttctagttataatttaaaatttggagagcatttttaacagtaattaatccagaggtggctcaaattgagtataagaattaagattatttaaaatactgcatgtctaccttctcggggatcatactttataacactttctgcttcagtagctcttcatagcttgccaagtatgctcccatattttctctctcgtgcctcgcaaatgaaagtcagataggctgggaactcatggggcagccctcagacttcaatgtgggcttcaaatccagtttcctgttctatatggtgctacatctttccagaaaatttccctcagagcccctcgccaaaacaaagcattattttgaccctgcatgctatttctttagctgtaggtgatagattagaacttctgtcagacatgttaatgacaaacataccaacagacaataaccaaagcaaatgtttccttcaagtgtgaaatgtgcaggggctcgtgggcaaggatgtattggcacactgtcctcttgaactgatagtgtcccaqcaatgttggaggttggcaccattcctggtccgacacttgaggacctgagagacatcaggtttagaatgagccaaagaaatcctacaagatggggagaattggtgtgcagcagcctaagtgttatagttaagtctaaagaagtatgaaagatcccctgtgttctctaaattgagcagaggggcctgcctaccaatatcactttttaggggactgaaccattgcaggttagacttggcttccaaagagtctgcctaagccaggggtggcagggtaggccatcatagctggatggcctcaaaagcagatgggggcagacttgccctcgtgatgccaggatttgagaggcagagtttctagagggagaccagtgctgcctctcacagtggcagttttttctctttgcaagaggaggggctgttcaattccatagaccagtgggcagatagccagttgaatactctgtgcatggtttgatcctttattagttcgctctaatatttttctgtagatccttttgtcctggactcaaaatctaatccatgcattgtatgataccgtagctctcctaaggtttgtgtttccttcaaaatgttttagttttcttcaactaaatttgatttttgctgttagaagtgacatatttttatggtatacactatgttccttttttctactgcgagtcaattttttgaattttcgtgagaaagaatatatctacaaattgcacgaaagtatcataaaaacagtactctagagcagcgctgtccaatagaaatataatctgagccacatgtataattttattttcttctagccacattaaagaagtaaaaagatacaagtagaactaattttaatgttttaattcagtatatccaaaatatcatttgaacatgtaattaatataaaattattaatgtgatattttacattcttttggtaatactagtcttcaaaatctggtatgtatcttacattgatagcacatctcactttgtactagccacattgcaagtgctcagtagccacatgtggctagtggctactgcactggacagcacagttctaggttccaccctaacacccaagtcctgtggattagaatcccagaatcagagctggaagtaaacatagagatcaaacctccttttaaaaatgaggacgctgaggcacagagtttaaatggcttgcatgaggtcatacagctaaattcagcctcaacagggtcttctgattccaggcactcttcccactccactacattactgtagtggtaattcttagggttaaaaaaagtgtagagtaggccgggcgcagtggctcatgcctgtaatcccagcactttgggaggccgaagtgggcggatcacgaggtcagg&gatcgagaccatcctggccaacatggtgaaaccccgtctctactgaaaatacaaagcaaaattagccaggtgtggtggcgggcgcctgtggtcccagctgctctggaggctgaggcagaatggcgtgaacccaggaggcagagatggcagtgagccaagatcgcgccactgcaccccagcctgggcgacagagcgagactccatctcaaaaaaaaaaaaaaaaaaaaaagaaaagaaaagaaaagtctagagaacattatattaagtggttattattgaagtagaccaaagtttataccataaggatatttttccttaaataccatgtttgaagaacaattatttattgatccttgaatctgtaagatcaaataacaagtctctatccatgttaccaaatttaaccttttgaaaataataaactttaaaatatcagatgtgttattacaggatgatacttggaatcaagtgaaatgagttatatggtcatcactaaatttagaaatctattgtgaaacaaagacaaacaggaaagtacagaatagagacttttagtaaataaatggaatttaaaagaaagtgtttatttacagtgtcacgacagaaaaggatgtctttgttgtcatagtctttgagggatctccgtaaaatctggggcacaggtacaagaaatagccaatatttagttcccagaccatgtttagtagtgtccagtttcagatcatgctgccaagaggtatctccccctcaggtgggtcatcactgagccctggaattggagactcatacttgcccagcacaatgttacgggcagacaggccgacatctatgattagctagaagccataaagaaaagctgctaagtggccactaggtgccacttttctgtttttgtaatgctttcattagcagatcttttttttccaagctccatggggcctatgagaggcatttatgatttttgtgcctacaataagtcagcctgtctggtgtgagttgttttatgagaaatgctttccaagggaggtctaggaagatcctgacacataagaactttggcttagagagctttccaggtgtagtgccaataaaaactgacctggaaagaaaacctgcccagcacggaacatgctttctgaactcacttgagagtgtatggtgtatgtcacttctcatatattcttgagtttagatttgtcttttatacaatttttagctcttttccagttcacttgtgctcgtctgtatattggtatttttaaatttttgtggtaaataatgaaaagagtgaaattatattttataattactcatttgtagtttttttttttaatttaataaacttcctccaaaaagtgctcccttaaaa

[0076] ARNT4 coding sequence in Table 7 is indicated by upper caseletters (nucieotides 220 to 2025) with-the termination codon underlined.

Diagnosis and Treatment of Circadian Rhythm Disorders

[0077] ARNT4 is involved in regulating circadian rhythm, e.g., byforming a heterodimer with Clock, a protein that regulates the timing offatigue and alertness. Individuals with circadian rhythm disorders arescreened for mutations in the an ARNT4 gene product or ARNT4 gene, e.g,.by detecting restriction fragment length polymorphisms (RFLPs) or byPCR. Individuals with symptoms of circadian rhythm disorders andidentified as having a mutated ARNT4 gene are treated by administeringDNA encoding a normal ARNT4 gene product. For example, DNA containingthe coding sequence of SEQ ID NO: 20 is administered to such individualsusing standard gene therapy techniques described herein. Similarly, anabnormally low or high level of ARNT4 protein or transcript is detectedin an individual suffering from such disorders, the levels can benormalized by antisense therapy to inhibit ARNT4 production or genetherapy to augment production. ARNT4 levels may also be altered toartificially regulate circadian rhythm, e.g., to induce long periods ofsleep in patients to improve the healing process or in individualstravelling long distances such astronauts during space travel.

Antisense Therapy

[0078] Nucleic acids complementary to all or part of the human EPAS1CDNA (GenBank Accession # U81984; SEQ ID NO: 7) may be used in methodsfor antisense treatment to inhibit expression of EPAS1. Nucleic acidscomplementary to all or part of the human ARNT4 cDNA (SEQ ID NO: 20) maybe used in methods for antisense treatment to inhibit expression ofARNT4. Antisense treatment may be carried out by administering to amammal, such as a human, DNA containing a promoter, e.g., an endothelialcell-specific promoter, operably linked to a DNA sequence (an antisensetemplate), which is transcribed into an antisense RNA. Alternatively, asmentioned above, antisense oligonucleotides may be introduced directlyinto vascular cells. The antisense oligonucleotide may be a shortnucleotide sequence (generally at least 10, preferably at least 14, morepreferably at least 20 (e.g., at least 30), and up to 100 or morenucleotides) formulated to be complementary to a portion, e.g., thecoding sequence, or all of EPAS1 MRNA or ARNT4 mRNA. For example, thesequence is complementary some or all of the C-terminal activationdomain; alternatively, the sequence may be complementary to all or partof the N-terminal DNA binding domain. The antisense sequence iscomplementary to DNA encoding residues 75 to 128, inclusive, of SEQ IDNO: 19; the antisense sequence. Alternatively, the antisense sequence iscomplementary to DNA encoding residues 155 to 207, inclusive, of SEQ IDNO: 19, or encoding residues 232 to 384 of SEQ ID NO: 19.,

[0079] Standard methods of administering antisense therapy have beendescribed (see, e.g., Melani et al., 1991, Cancer Res. 51:2897-2901).Following transcription of a DNA sequence into an antisense RNA, theantisense RNA binds to its target nucleic acid molecule, such as an MRNAmolecule, thereby inhibiting expression of the target nucleic acidmolecule. For example, an antisense sequence complementary to a portionor all of EPAS1 MRNA could be used to inhibit the expression of EPAS1,thereby decreasing the level of transcription of angiogenic factors suchas VEGF or VEGF-Rs, which in turn leads to a decrease in new bloodvessel formation. Oligonucleotides complementary to various portions ofEPAS1 mRNA or ARNT4 MRNA can readily be tested in in vitro for theirability to decrease production of their respective gene products, usingassays similar to those described herein. Sequences which decreaseproduction of EPAS1 message or ARNT4 message in vitro cell-based orcell-free assays can then be tested in vivo in rats or mice to determinewhether blood vessel formation is decreased.

[0080] Preferred vectors for antisense templates are viral vectors,including those derived from replication-defective hepatitis viruses(e.g., HBV and HCV), retroviruses (see, e.g., WO 89/07136; Rosenberg etal., 1990, N. Eng. J. Med. 323(9):570-578), adenovirus (see, e.g.,Morsey et al., 1993, J. Cell. Biochem., Supp. 17E,), adeno-associatedvirus (Kotin et al., 1990, Proc. Natl. Acad. Sci. USA 87:2211-2215,),replication defective herpes simplex viruses (HSV; Lu et al:, 1992,Abstract, page 66, Abstracts of the Meeting on Gene Therapy, Sept.22-26, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), and anymodified versions of these vectors. The invention may utilize any otherdelivery system which accomplishes in vivo transfer of nucleic acidsinto eucaryotic cells. For example, the nucleic acids may be packagedinto liposomes, receptor-mediated delivery systems, non-viral nucleicacid-based vectors, erythrocyte ghosts, or microspheres (e.g.,microparticles; see, e.g., U.S. Pat. No. 4,789,734; U.S. Pat. No.4,925,673; U.S. Pat. No. 3,625,214; Gregoriadis, 1979, Drug Carriers inBiology and Medicine, pp. 287-341 (Academic Press,). Alternatively,naked DNA may be administered. Delivery of nucleic acids to a specificsite in the body for antisense therapy may also be accomplished using abiolistic delivery system, such as that described by Williams et al.,1991, Proc. Natl. Acad. Sci. USA 88:2726-2729.

[0081] Antisense oligonucleotides may consist of DNA, RNA, or anymodifications or combinations thereof. As an example of themodifications that the oligonucleotides may contain, inter-nucleotidelinkages other than phosphodiester bonds, such as phosphorothioate,methylphosphonate, methylphosphodiester, phosphorodithioate,phosphoramidate, phosphotriester, or phosphate ester linkages (Uhlman etal., 1990, Chem. Rev. 90(4) :544-584; Anticancer Research, 1990,10:1169) may be present in the oligonucleotides, resulting in theirincreased stability. oligonucleotide stability may also be increased byincorporating 3′-deoxythymidine or 2′-substituted nucleotides(substituted with, e.g., alkyl groups) into the oligonucleotides duringsynthesis, by providing the oligonucleotides as phenylisoureaderivatives, or by having other molecules, such as aminoacridine orpoly-lysine, linked to the 3′ ends of the oligonucleotides e.g.,Anticancer Research, 1990, 10:1169-1182). Modifications of the RNAand/or DNA nucleotides may be present throughout the oligonucleotide, orin selected regions of the oligonucleotide, e.g., in the 5′ and/or 3′ends. The antisense oligonucleotides may also be modified so as toincrease their ability to penetrate the target tissue by, e.g., couplingthe oligonucleotides to lipophilic compounds. Antisense oligonucleotidesbased on the human EPAS1 nucleotide sequence (SEQ ID NO: 7) or the humanARNT4 nucleotide sequence (SEQ ID NO: 20) can be made by any methodknown in the art, including standard chemical synthesis, ligation ofconstituent oligonucleotides, and transcription of DNA complementary tothe all or part of the EPASI CDNA or ARNT4 CDNA.

[0082] EPAS1 is naturally expressed in vascular endothelial cells. Thesecells are, therefore, the preferred cellular targets for antisensetherapy. Targeting of antisense oligonucleotides to endothelial cells isnot critical to the invention, but may be desirable in some instances,e.g. systemic administration of antisense compositions. Targeting may beachieved, for example, by coupling the oligonucleotides to ligands ofendothelial cell surface receptors. Similarly, oligonucleotides may betargeted to endothelial cells by being conjugated to monoclonalantibodies that specifically bind to endothelial-specific cell surfaceproteins. Antisense compositions may also be administered locally, e.g.,at the site of an atherosclerotic lesion or at the site of a tumor.

[0083] Therapeutic applications of antisense oligonucleotides in generalare described, e.g., in the following review articles: Le Doan et al.,Bull. Cancer 76:849-852, 1989; Dolnick, Biochem. Pharmacol. 40:671-675,1990; Crooke, Annu. Rev. Pharmacol. Toxicol. 32, 329-376, 1992.Antisense nucleic acids may be used alone or combined with one or morematerials, including other antisense oligonucleotides or recombinantvectors, materials that increase the biological stability of theoligonucleotides or the recombinant vectors, or materials that increasethe ability of the therapeutic compositions to penetrate endothelialcells selectively.

[0084] Therapeutic compositions, e.g., inhibitors of EPAS1 and/or ARNT4transcription or transactivating function, may be administered inpharmaceutically acceptable carriers (e.g., physiological saline), whichare selected on the basis of the mode and route of administration andstandard pharmaceutical practice. Suitable pharmaceutical carriers, aswell as pharmaceutical necessities for use in pharmaceuticalformulations, are described in Remington's Pharmaceutical Sciences, astandard reference text in this field, and in the USP/NF. The compoundmay be administered with intravenous fluids as well as in combinationwith other anti-inflammatory agents, e.g., antibiotics; glucocorticoids,such as dexamethasone (Dex), or other chemotherapeutic drugs for thetreatment of atherosclerotic lesions and tumors, respectively.

[0085] A therapeutically effective amount is an amount which is capableof producing a medically desirable result in a treated animal. As iswell known in the medical arts, dosage for any one patient depends uponmany factors, including the patient's size, body surface area, age, theparticular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently. Dosages will vary, but a preferred dosage for intravenousadministration of DNA is approximately 10⁶ to 10²² copies of the DNAmolecule. The compositions of the invention may be administered locallyor systemically. Administration will generally be parenterally, e.g.,intravenously. As mentioned above, DNA may also be administered directlyto the target site, e.g., by biolistic delivery to an internal orexternal target site or by catheter to a site in an artery.

Gene Therapy

[0086] Compositions which enhance intracellular production of EPAS1 (orits binding to a cis-acting regulatory region of a gene encoding VEGF ora VEGF-R) or ARNT4 may be used in methods to promote new blood vesselformation, e.g., to promote angiogenesis in wound healing (e.g., healingof broken bones, burns, diabetic ulcers, or traumatic or surgicalwounds) and organ transplantation. Such compounds may be used to treatperipheral vascular disease, cerebral vascular disease, hypoxic tissuedamage (e.g., hypoxic damage to heart tissue), or coronary vasculardisease as well as to treat patients who have, or have had, transientischemic attacks, vascular graft surgery, balloon angioplasty,frostbite, gangrene, or poor circulation.

[0087] Since EPAS1 and ARNT4 are nuclear proteins, a preferred method ofincreasing the levels of these proteins or polypeptides in a cell (toincrease transcription of such angiogenic factors as VEGF or VEGF-Rs) isintracellular expression of recombinant EPAS1 or ARNT4 or activefragments thereof, e.g., transactivating fragments. DNA encoding EPAS1or ARNT4 is administered alone or as part of an expression vector asdescribed above. The DNA introduced into its target cells, e.g.,endothelial cells at an anatomical site in need of angiogenesis, directsthe production of recombinant EPAS1 or ARNT4 or fragments thereof in thetarget cell, to enhance production of new blood vessels. For inhibitionof angiogenesis, gene therapy are also used to introduce administer DNAencoding a dominant negative mutant of EPAS1 such as DNA encoding apolypeptide with the amino acid sequence of SEQ ID NO: 4 or apolypeptide with the amino acid sequence of residues 486-639 of SEQ IDNO: 6.

Antibodies and Intrabodies

[0088] Anti-EPAS1 antibodies were obtained using techniques well knownin the art. Such antibodies can be polyclonal or monoclonal. Polyclonalantibodies can be obtained, for example, by the methods described inGhose et al., Methods in Enzymology, Vol. 93, 326-327, 1983. An EPAS1polypeptide, or an antigenic fragment thereof, was used as the immunogento stimulate the production of EPAS1-reactive polyclonal antibodies inthe antisera of animals such as rabbits, goats, sheep, rodents and thelike. EPAS1-specific antibodies were raised by immunizing animals with aC-terminal EPAS1 polypeptide spanning amino acids 668-829 of human EPAS(PGGSTSHLMWKRMKNLRGGSCPLMPDKPLSANVPNDKFTQNPMRGLHPLRHLPLPQPPSAISPGENSKSRFPPQCYATQYQDYSLSSAHKVSGMASRLLGP; (SEQ ID NO: 17)and a C-terminal EPAS polypeptide spanning amino acids 641-875 of mouseEPAS1 DPPLHFGPTKWPVGDQSAESLGALPVGSWQLELPSAPLHVSMFKMRSAKDFGARGPYMMSPAMIALSNKLKLKRQLEYEEQAFQDTSGGDPPGTSSSHLMWKRMKSLMGGTCPLMPDKTISANMAPDEFTQKSMRGLGQPLRHLPPPQPPSTRSSGENAKTGFPPQCYASQFQDYGPPGAQKVSGVASRLLGPSFEPYLLPELTRYDCEVNVPVPGSST LLQGRDLLRALDQAT (SEQID NO: 18).

[0089] Monoclonal antibodies are obtained by the process described byMilstein and Kohler in Nature, 256:495-97, 1975, or as modified byGerhard, Monoclonal Antibodies, Plenum Press, 1980, pages 370-371.Hybridomas are screened to identify those producing antibodies that arehighly specific for an EPAS1 polypeptide. Preferably, the antibody willhave an affinity of at least about 10⁸ liters/mole and more preferably,an affinity of at least about 10⁹ liters/mole. Monoclonal antibodies canbe humanized by methods known in the art, e.g, MAbs with a desiredbinding specificity can be commercially humanized (Scotgene, Scotland;Oxford Molecular, Palo Alto, Calif.).

[0090] Following identification of a hybridoma producing a suitablemonoclonal antibody, DNA encoding the antibody is cloned. DNA encoding asingle chain EPAS1-specific antibody in which heavy and light chainvariable domains (separated by a flexible linker peptide such asGly₄-Ser₃ (SEQ ID NO: 16) is cloned into an expression vector usingknown methods (e.g., Marasco et al., 1993, Proc. Natl. Acad. Sci. USA90:7889-7893 and Marasco et al., 1997, Gene Therapy 4:11-15) Suchconstructs are introduced into cells, e.g., using gene therapytechniques described herein, for intracellular production of theantibodies. Intracellular antibodies, i.e., intrabodies, are used toinhibit binding of endogenous EPAS1 to its target DNA (e.g., cis-actingregulatory sequences of genes encoding VEGF or VEGF-Rs), which in turn,decreases production of these angiogenic factors and decreases new bloodvessel formation in the treated mammal. Intrabodies which bind to aC-terminal transactivation domain of EPAS1 inhibit the ability of EPAS1to induce transcription of a gene encoding an angiogenic factor such asVEGF or a VEGF-R. A similar strategy is used to make intrabodies whichbind to intracellular ARNT4. Such intrabodies bind to ARNT4 and preventheterodimeriation with EPAS1, and as a result, inhibit transactivationof the VEGF promoter. Inhibition of VEGF promoter activity, in turn,leads to inhibition of new blood vessel formation.

[0091] Other embodiments are within the following claims.

What is claimed is:
 1. A method of inhibiting angiogenesis in a mammalcomprising administering to said mammal a compound which inhibitsbinding of endothelial PAS domain protein-1 (EPAS1) to cis-actingtranscription regulatory DNA of an angiogenic factor.
 2. The method ofclaim 1, wherein said angiogenic factor is a vascular endothelial growthfactor receptor (VEGF-R).
 3. The method of claim 2, wherein saidreceptor is KDR/flk-1.
 4. The method of claim 2, wherein said receptoris flt-1.
 5. The method of claim 1, wherein said angiogenic factor isvascular endothelial growth factor (VEGF).
 6. The method of claim 1,wherein said angiogenic factor is Tie2.
 7. The method of claim 1,wherein said compound inhibits transcription of said angiogenic factor.8. The method of claim 1, wherein said regulatory DNA comprisesGCCCTACGTGCTGTCTCA (SEQ ID NO:1).
 9. The method of claim 1, wherein saidcompound is an EPAS1 polypeptide lacking a transactivation domain. 10.The method of claim 9, wherein said transactivation domain comprises theamino acid sequence of SEQ ID NO:
 2. 11. The method of claim 9, whereinsaid transactivation domain comprises the amino acids 486-639 of SEQ IDNO:
 6. 12. The method of claim 9, wherein said polypeptide comprises theamino acid sequence of SEQ ID NO:
 4. 13. The method of claim 1, whereinsaid compound is a nucleic acid encoding an EPAS1 polypeptide lackingthe amino acid sequence of SEQ ID NO:
 2. 14. The method of claim 1,wherein said compound is a nucleic acid encoding an EPAS1 polypeptidelacking amino acids 486-639 of SEQ ID NO:
 6. 15. The method of claim 1,wherein said compound is a antisense nucleic acid molecule comprising atleast 10 nucleotides, wherein the sequence of said molecule iscomplementary to part of or all ofan MRNA encoding EPAS1 polypeptide.16. The method of claim 1, wherein said compound is an EPAS1-specificintrabody.
 17. The method of claim 1, wherein said compound isadministered to a site of an atherosclerotic lesion in said mammal. 18.The method of claim 1, wherein said compound is administered to a tumorsite in said mammal.
 19. An antibody which binds to EPAS1 .
 20. Theantibody of claim 19, wherein said antibody binds to a C-terminalactivation domain of EPAS1.
 21. The antibody of claim 20, wherein saidactivation domain comprises SEQ ID NO:
 2. 22. A method of promotingangiogenesis in a mammal comprising administering to said mammal acompound which increases expression of VEGF or a VEGF-R in anendothelial cell.
 23. The method of claim 19, wherein said VEGF-R isKDR/flk-1 or flt-1.
 24. A substantially pure DNA comprising a sequenceencoding a aryl hydrocarbon receptor nuclear translocator-4 (ARNT4)polypeptide.
 25. The DNA of claim 24, wherein said DNA encodes a humanARNT4 polypeptide.
 26. The DNA of claim 24, wherein said polypeptidecomprises the amino acid sequence of residues 75 to 128, inclusive, ofSEQ ID NO:
 19. 27. The DNA of claim 24, wherein said polypeptidecomprises the amino acid sequence of residues 155 to 207, inclusive, ofSEQ ID NO:
 19. 28. The DNA of claim 24, wherein said polypeptidecomprises the amino acid sequence of residues 232 to 384, inclusive, ofSEQ ID NO:
 19. 29. A substantially pure DNA comprising a nucleotidesequence having at least 50% sequence identity to SEQ ID NO:
 20. 30. TheDNA of claim 24, wherein said DNA comprises the coding sequences of SEQID NO:
 20. 31. The DNA of claim 24, wherein said polypeptide comprisesthe amino acid sequence of SEQ ID NO:
 19. 32. A substantially pure DNAcomprising a strand which hybridizes at high stringency to a strand ofDNA consisting of the coding sequence of SEQ ID NO: 20, or thecomplement thereof.
 33. A substantially pure DNA comprising a sequencesat least 50% sequence identity to the coding sequence of SEQ ID NO: 20,and encoding a polypeptide having the biological activity of an ARNT4polypeptide.
 34. A substantially pure ARNT4 polypeptide.
 35. Thepolypeptide of claim 34, wherein said polypeptide is human ARNT4. 36.The polypeptide of claim 34, wherein said polypeptide comprises theamino acid sequence of residues 75 to 128, inclusive, of SEQ ID NO: 19.37. The polypeptide of claim 34, wherein said polypeptide comprises theamino acid sequence of residues 155 to 207, inclusive, of SEQ ID NO: 19.38. The polypeptide of claim 34, wherein said polypeptide comprises theamino acid sequence of residues 232 to 384, inclusive, of SEQ ID NO: 19.39. The polypeptide of claim 34, wherein said polypeptide comprises theamino acid sequence of SEQ ID NO:
 19. 40. The polypeptide of claim 34,wherein said polypeptide comprises an amino acid sequence at least 50%identical to SEQ ID NO:
 19. 41. The polypeptide of claim 34, whereinsaid polypeptide comprises the amino acid sequence of SEQ ID NO:
 19. 42.A vector comprising the DNA of claim
 24. 43. A host cell comprising theDNA of claim
 24. 44. A transgenic non-human animal the germ cells andnucleated somatic cells of which comprise a null mutation in a geneencoding ARNT4.
 45. A method of inhibiting angiogenesis in a mammalcomprising administering to said mammal a compound which inhibitsbinding of EPAS1 to ARNT4.
 46. The method of claim 45, wherein saidcompound is an ARNT4 polypeptide.
 47. The method of claim 45, whereinsaid polypeptide comprises the amino acid sequence of residues 75 to128, inclusive, of SEQ ID NO:
 19. 48. The method of claim 45, whereinsaid polypeptide comprises the amino acid sequence of residues 155 to207, inclusive, of SEQ ID NO:
 19. 49. The method of claim 45, whereinsaid polypeptide comprises the amino acid sequence of residues 232 to384, inclusive, of SEQ ID NO:
 19. 50. The method of claim 45, whereinsaid polypeptide comprises the amino acid sequence of SEQ ID NO:
 19. 51.An EPAS1 polypeptide lacking a transactivation domain.
 52. Thepolypeptide of claim 51, wherein said transactivation domain comprisesthe amino acid sequence of SEQ ID NO:
 2. 53. The polypeptide of claim51, wherein said transactivation domain comprises the amino acids486-639 of SEQ ID NO:
 6. 54. The polypeptide of claim 51, wherein saidpolypeptide comprises the amino acid sequence of SEQ ID NO:
 4. 55. Anucleic acid encoding an EPAS1 polypeptide lacking the amino acidsequence of SEQ ID NO:
 2. 56. The nucleic acid of claim 55, wherein saidnucleic acid encodes an EPASI polypeptide lacking amino acids 486-639 ofSEQ ID NO: 6.